UNC-45 assisted myosin folding depends on a conserved FX3HY motif implicated in Freeman Sheldon Syndrome

Myosin motors are critical for diverse motility functions, ranging from cytokinesis and endocytosis to muscle contraction. The UNC-45 chaperone controls myosin function mediating the folding, assembly, and degradation of the muscle protein. Here, we analyze the molecular mechanism of UNC-45 as a hub in myosin quality control. We show that UNC-45 forms discrete complexes with folded and unfolded myosin, forwarding them to downstream chaperones and E3 ligases. Structural analysis of a minimal chaperone:substrate complex reveals that UNC-45 binds to a conserved FX3HY motif in the myosin motor domain. Disrupting the observed interface by mutagenesis prevents myosin maturation leading to protein aggregation in vivo. We also show that a mutation in the FX3HY motif linked to the Freeman Sheldon Syndrome impairs UNC-45 assisted folding, reducing the level of functional myosin. These findings demonstrate that a faulty myosin quality control is a critical yet unexplored cause of human myopathies.


Introduction
Muscle contraction relies on the sliding of actin and myosin filaments against each other. For efficient power generation, the two myofilaments are arranged in a quasicrystalline manner in the sarcomere, the basic building block of all muscle cells 1 .
Maintaining the higher-order organization of the sarcomere during exercise, proteotoxic stress, and ageing is of utmost importance, as reflected by the many myopathies connected with sarcomeric components 2 . To prevent damage and to oversee the highly specialized proteome of muscle cells, an extended set of molecular chaperones is employed 3 . Abundance and functional diversity of the involved folding factors reflect the intricate structures and assembly pathways of muscle proteins, such as myosin 3,4 . Notably, despite containing an ATPase domain that poses grand challenges for the cellular chaperone machinery 5,6 , myosin is the most abundant protein in muscle cells, accounting for about 16% of the entire proteome 7 .
Both the folding of myosin and the formation of thick filaments rely on the activity of multiple assembly factors 8 , including members of the general chaperone machinery (e.g. HSP70, HSP90 and TRiC), but also muscle cell-specific chaperones. Genetic screens have identified UNC-45 as an essential regulator of myosin proteostasis [9][10][11][12] .
In addition to functioning as a folding factor of the myosin motor, UNC-45 can form linear protein chains that provide regular spaced docking sites for HSP70 and HSP90. This molecular assembly line allows for coordinated chaperone activity on myosin heads protruding from the thick filament 13 . The important role of UNC-45 for myosin filament formation and sarcomere integrity is reflected by detrimental phenotypes upon genetic disruption. Knock-out of UNC-45 results in embryonic lethality, and both its upregulation and downregulation are linked to severe sarcomere defects 14,15 . Consistent with these findings, UNC-45 mutations have been directly linked to human myopathies 16,17 .
UNC-45, which is present in all eukaryotes, employs a UCS domain to interact with myosin, a central domain that mediates oligomerization, and a TPR domain that interacts with members of the protein quality control (PQC) network 18 . The HSP70/HSP90 chaperones bind to the TPR-domain and are required for myosin assembly during muscle development 11,19 . In addition to coordinating filament formation, UNC-45 maintains the level of functional myosin in mature muscle cells . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 9, 2022. ; https://doi.org/10.1101/2022.08.08.502945 doi: bioRxiv preprint 4 during stress situations. To fulfill this role, UNC-45 uses its TPR domain to interact with the ubiquitin ligase UFD-2, enabling the E3 enzyme to selectively target and ubiquitinate aberrant myosin molecules 20 . Consistent with this quality control function, stress situations applied to muscle tissues induce the interaction of UNC-45 with already assembled myosin molecules 21 , whereas knockdown of UNC-45 results in an increase of myosin levels in the sarcomere 22 . However, the mechanism by which the chaperone distinguishes between folding competent and severely damaged myosin proteins and how it channels these into folding and degradation pathways is not understood. Moreover, and in contrast to the well-characterized general chaperones HSP70, HSP90, and TRIC [23][24][25][26] , little is known about the substrate targeting mechanism of specialized folding factors such as UNC-45. Therefore, studying the interplay between UNC-45 and myosin is not only important to better understand how triage decisions are made in the PQC system but also to delineate the mode of action of client-specific chaperones.
By pursuing an integrative structural biology approach, we reveal that UNC-45 is able to bind myosin present in different conformations. Once bound to UNC-45, the fate of the myosin substrate depends on its folding state. We were able to delineate this mechanism by separating discrete UNC-45 complexes with "folding-competent" or "degradation-prone" myosin, and determining the high-resolution crystal structure of a minimal chaperone:substrate complex. We identified the conserved FX 3 HY myosin motif as primary site of UNC-45 interaction and applied cellular assays to demonstrate that this myosin epitope is essential for UNC-45 recruitment and maturation of the muscle protein. Strikingly, a Y582S mutation in this motif is linked to a severe developmental myopathy, the Freeman Sheldon Syndrome (FSS) 27,28 . The site-specific mutation abrogates the interaction between UNC-45 and myosin, causing severe protein misfolding in cells. These data directly relate myosin PQC with myosin-based myopathies, a connection that has not been described and explored so far.
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UNC-45 forms separate complexes with folded and unfolded myosin
To characterize the interplay between chaperone and substrate at a molecular level, we reconstituted the complex between C. elegans UNC-45 and the motor domain of MHC-B muscle myosin (myo). After co-expression in insect cells, the UNC-45:myo complex was purified by tandem affinity chromatography and size exclusion chromatography (SEC) 20 . In the final SEC run, the UNC-45:myo complex eluted in a reproducible series of high-and low-molecular weight species (Fig. 1a). Since the stoichiometry of UNC-45 and myosin was similar in all fractions, the elution peaks likely reflect different conformational states of the bound myosin. To address this point, we compared the SEC elution profiles with those of folded and heat-denatured myosin alone (Supp. Fig. 1). This comparison suggested that the high-molecular weight peak should represent UNC-45 bound to unfolded myosin (UNC-45:myo UF ), whereas the second peak contains the chaperone engaged with a mostly folded substrate (UNC-45:myo F ). To corroborate the folding state of bound myosin, we took advantage of the lower stability of myosin compared to UNC-45. Myosin, but not UNC-45, unfolds at 27°C allowing us to selectively destabilize myosin at elevated temperature 29 . We thus incubated the two complexes at 4°C and 27°C and reapplied the samples to SEC analysis. For UNC-45:myo UF , the elution volume remained almost unchanged after heat treatment, suggesting that the UNC-45-bound myosin is already unfolded (Fig. 1b). In contrast, when the UNC-45:myo F complex was incubated at 27°C, the reapplied complex shifted to an earlier elution volume, characteristic of the unfolded myosin (Fig. 1c). These data strongly support our notion that myosin in the UNC-45:myo F complex is present in a mostly folded conformation, which is sensitive to heat-induced unfolding.
To identify interacting regions of chaperone and substrate, we performed crosslinking mass spectrometry (XLMS) experiments. Analysis of the UNC-45:myo F complex containing the folded client protein indicated that UNC-45 interlinks can be grouped into two classes (Fig. 1d Supp. Fig. 2). They either comprise residues of predominantly disordered UNC-45 regions (class-I: neck-domain loop 508-524; UCSloop 602-630; UCS C-terminus 931-961) or residues that are part of structurally welldefined sub-domains (class-II). While class I contacts showed a promiscuous crosslinking pattern, the central part of the UCS-domain (residues 695-720) is . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 9, 2022. ; https://doi.org/10.1101/2022.08.08.502945 doi: bioRxiv preprint 6 involved in specific class-II contacts via its structurally defined ARM-repeats.
Crosslinked residues border the groove of the UCS domain that, based on structural alignment with β -catenin 13 , has been suggested to form a myosin-binding canyon.
We also observed specific class-II interlinks to the TPR domain of UNC-45, hinting to an additional myosin binding site. These data are consistent with an HSP70/HSP90 independent role of the TPR domain in yielding functional myosin 29 . Class-II crosslinks with myosin map to an N-terminal region (residues 30-150) adjacent to the SH3-like domain, residues 550-598 of the lower 50 kDa subdomain and the Cterminal part of the myosin motor adjacent to the converter region (Fig. 1d).
Importantly, most interlinks are located on the same face of the myosin molecule, extending from the actin-binding site to the SH3-domain (Fig. 1e). The defined crosslinking pattern between UNC-45 and MHC-B suggests that the two proteins bind to each other in a structurally defined manner as expected for folded proteins.
Contrary, in the UNC-45:myo UF complex, UNC-45 is engaged in many unspecific interlinks with myosin (Fig. 1d). When plotting these interlinks, the unfolded nature of myosin is reflected by continuous streaks of dots (Supp, Fig. 2), suggesting that the relevant residues are part of disordered segments that arbitrarily interact with nearby sites. Moreover, the contacts are not restricted to clearly defined regions of myosin but rather involve all subdomains. In conclusion, the comparative SEC and XLMS analysis indicate that UNC-45 is capable of binding its substrate in folded and unfolded conformations, yielding functionally different chaperone:substrate complexes.

The folding state of myosin determines its interactions with PQC factors
To investigate whether the folding state of the captured substrate influences the channeling into folding and degradation pathways, we tested how the UNC-45:myo complexes interact with the molecular chaperone HSP90 and the UFD-2 ubiquitin ligase, respectively. Owing to the binding of HSP90 to the UNC-45 TPR domain 29 , we performed interaction assays with the K82E mutant of UNC-45. As this mutant is known to abrogate TPR-dependent binding 13 , it allows to selectively monitor the interaction of HSP90 chaperone with the captured myosin. Upon co-expression in insect cells, UNC-45 K82E :myo complexes isolated by tandem affinity chromatography showed a similar SEC profile as the complexes with wildtype UNC-45, being engaged with myo F and myo UF (Fig. 2a). In pull-down assays, HSP90 bound to both . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 9, 2022. ; https://doi.org/10.1101/2022.08.08.502945 doi: bioRxiv preprint 7 UNC-45 K82E complexes containing either intact or unfolded myosin (Fig. 2a). This finding is consistent with HSP90 playing a dual role in maintaining the functionality of already folded and assembled myosin molecules in muscle sarcomeres 21 as well as serving as an E3 ligase co-factor in protein degradation pathways.
We next addressed the interplay between UNC-45, myosin and UFD-2, the ubiquitin ligase implicated in myosin quality control 20 . To map interaction sites between substrate, adaptor and E3 ligase, we pursued an XLMS approach. The three proteins were mixed in stoichiometric amounts and incubated at 4°C or 27°C, with the higher temperature selectively destabilizing myosin. Only when incubated at 27°C, a functional complex between UNC-45, myo UF and UFD-2 was formed (Fig. 2b), Recently it has been proposed that UFD-2 can interact with the TPR domain of CHN-1 and stimulate its E3 ligase activity through an internal EEYD motif near the catalytic Ubox domain 30 (Supp Fig. 3b). Our XLMS data of the UFD-2/UNC-45/myosin complex indicates that UFD-2 also interacts with the UNC-45 TPR domain, however rather through an uncharacterized motif in the center of the ubiquitin ligase (residues 271-322, Fig. 2c, Supp. Fig. 3b), pointing to a different binding mode. Interestingly, UFD-2 engages the myosin substrate in a specific manner, as evidenced by crosslinks between its catalytic Ubox module and the terminal segments of the myosin motor domain (Fig. 2c). To test the functional relevance of the intercrosslinks, we performed in vitro ubiquitination assays and mapped the ubiquitination sites on myosin. We observed that at higher temperature, when myosin is destabilized and complex formation is favored, the ubiquitination reaction was markedly enhanced (Supp. Fig. 3a), confirming the selective targeting of misfolded myosin. Mapping the ubiquitination sites of myo UF revealed that UFD-2 preferentially targets lysine residues in the N-and C-terminal regions of the motor domain, as predicted by the XLMS analysis (Fig. 2d). We thus propose that the central portion of the myosin motor, recognized and bound by UNC-45, is present in a mostly unfolded conformation enabling the N-and C-terminal segments to access the Ubox of the E3 enzyme. To further evidence the preference of UFD-2 for targeting an unfolded substrate, we performed in vitro ubiquitination assays with natively purified chaperone:substrate complexes and assessed myosin ubiquitination over time ( Fig. 2e-f, Supp. Fig. 3c). As predicted, UFD-2 showed a clear preference for ubiquitinating myosin contained in the UNC-45:myo UF complex, whereas the reaction was slower and less efficient for UNC-45:myo F . In sum, our data show that UNC-45 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 9, 2022. Δ loop variants, we observed several non-specific hits, however, they did not bind to the 547-588 motif recognized by wildtype UNC-45 (Fig. 3a). Notably, the 547-588 stretch corresponds to a β -hairpin in the motor domain that is engaged in most class-II interlinks with UNC-45 in the XLMS datasets (Fig. 1d, Fig. 3b).
To confirm that the identified myosin stretch mediates UNC-45 binding in a specific manner, we characterized this interaction by thermal shift assays. In this approach, the stability of a protein -reflected by the melting temperature T m -is measured in the presence of ligands, assuming that ligand binding increases protein stability. To control for non-specific binding events, we used the UNC-45 Δ UCS mutant and a peptide with a randomly scrambled sequence of the myosin motif (Fig. 3c, Supp. Fluorescence anisotropy measurements, which were carried out in a qualitative manner due to low affinity of the binder, confirmed the specific interaction between the identified myosin ligand and UNC-45 (Supp. Fig. 4b). In sum, these data indicate that residues 547-588 of the myosin motor domain contain the primary interaction site for UNC-45.
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Structure of minimal UNC-45:myosin complex reveals FX 3 HY motif
To investigate the structural basis of myosin recognition, we pursued a crystallographic approach. Given the difficulties in obtaining a co-crystal structure with C. elegans UNC-45, we moved to the less complex fungal orthologue SHE4.
Despite lacking the TPR-domain, SHE4 contains a functional UCS-domain for myosin binding 13,32 . Moreover, sequence alignments indicated that the myosin motif recognized by UNC-45 is conserved in fungal isoforms (Fig. 4a) and has been previously postulated to be part of the SHE4 binding motif 32 . Bioinformatic analysis revealed that Kluyveromyces lactis SHE4 is the most promising candidate for crystallographic studies, having a functional UCS domain but lacking various flexible, non-conserved regions that impeded crystallization of the yeast ortholog 32 (Supp. To characterize the structural motif recognized by the UCS domain, we synthesized a series of Myo4 peptides, encompassing the β -hairpin motif 547-588 identified in the peptide spot assay. Isothermal titration calorimetry (ITC) measurements showed that 13 residues (561-573) comprise the core of the myosin motif, binding to SHE4 KL with a K D of 0.6 μ M (Fig. 4b). After intense co-crystallization trials with various myosin peptides, we obtained a well-diffracting SHE4 co-crystal with a peptide comprising Myo4 residues 561-581. We determined the co-crystal structure at a resolution of 2.4 Å and were able to visualize a minimal SHE4:Myo4 complex in atomic detail. In this structure, the Myo4 residues Lys561-Phe562-Ile563-Val564-Ser565-His566-Tyr567 were well-defined by electron density (Fig. 4e). The eight myosin residues are accommodated in the UCS-canyon by a dense network of hydrogen bonds and hydrophobic interactions. The tight binding mode forces the Myo4 peptide to adopt a . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 9, 2022. ; https://doi.org/10.1101/2022.08.08.502945 doi: bioRxiv preprint bent conformation, with its C-terminal portion being directed out of the central groove of the UCS-domain. Notably, this bent conformation is distinct from the β -turn observed in the native myosin structure, preventing UNC-45 to target functional myosin (Supp. Fig. 6). As for its substrate, the interacting residues in the UCS domain of SHE4 are conserved across species (Supp. Fig. 7) suggesting that the observed binding mode is generally relevant. To validate the structural data, we mutated the strictly conserved Asn475 that is located in the UCS binding groove and binds to the backbone of the captured myosin peptide (Fig. 4e, Supp. Fig. 7). ITCmeasurements showed that binding to the myosin peptide was abrogated in the N475A mutant, confirming its participation in substrate binding (Fig. 4c).
Given the conservation of the myosin residue Phe577, His581 and Tyr582, which are accommodated in specific pockets of the UCS canyon ( Fig. 4a-d), we tested the general relevance of these residues in the C. elegans system. Since UNC-45 precipitated during ITC measurements, we performed thermal shift measurements, incubating UNC-45 with the respective MHC-B peptide. Contrary to the native peptide that elevated UNC-45 stability by 5.5°C, introducing the F577A, H581A or Y582A mutations only resulted in a minor increase in T m (Supp. Fig. 8a). Consistently, fluorescence anisotropy measurements supported the crucial role of these residues in mediating UNC-45 binding, as F577A, H581A and Y582A myosin peptides were strongly impaired in their interaction with UNC-45 (Fig. 5a). Furthermore, the binding of this motif to UNC-45 fits well to our crosslinking data (Fig. 1d), according to which myosin residues 576-587 are accommodated in the center of the UCS canyon, juxtaposing the adjacent motor domain regions to the UNC-45 chaperone. The observed crosslinks suggest that the complex is held together on one side by links from myosin residues Lys550 and Lys558, and on the other side by a link from residue Tyr588 (Fig. 3b). In concert, these interactions yield a chaperone-substrate complex with a well-defined topology that locks the β -hairpin motif in place. Taken together, our structural and biochemical data indicate that the conserved myosin residues Phe577, His581 and Tyr582 mediate binding to C. elegans UNC-45. We thus propose that the respective FX 3 HY motif represents a universal and highly specific substrate epitope, recognized by the myosin-specific chaperone UNC-45.
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Disruption of the FX 3 HY motif abolishes myosin folding in the cell
To test the functional relevance of the FX 3 HY-motif in a cellular context we monitored UNC-45 dependent maturation of myosin in insect cells 29 . Since UNC-45 is essential to produce soluble myosin, UNC-45/myosin co-expression can be used as a proxy for evaluating chaperone-substrate interactions. By assessing the yields of soluble myosin produced in insect cells, we found that replacing the β -hairpin loop (581-HYAG-584) with a GSGS spacer impairs UNC-45-dependent myosin folding (Fig. 5b). Strikingly, the same deleterious effect was observed for the single-site mutants F577A, H581A or Y582A (Fig. 5b-c), highlighting the impact of the FX 3 HY motif on myosin maturation. We then looked for disease causing mutations in this motif and found that the Freeman-Sheldon Syndrome (FSS), a myopathy associated with severe multiple congenital contracture 34 , is linked to the substitution of a serine at the conserved Tyr582 site. To explore whether the Y582S mutation may impact interaction with UNC-45, we co-expressed the respective MHC-B variant with UNC-45 in insect cells and observed that this mutation significantly decreased the levels of soluble myosin (Fig. 5c). Fluorescence anisotropy measurements with a myosin peptide carrying the Y582S mutation confirmed the in vivo data, as binding to UNC-45 was markedly reduced (Fig. 5a). The resultant misfolding of myosin may underlie the phenotype observed in a Drosophila melanogaster FSS disease model, in which the Y582S mutation led to a progressive disruption of muscle sarcomeres, although the kinetic properties of the isolated mutant were more similar to wildtype myosin when compared to other FSS mutants 27 . Contrary to the Y582S mutation, introducing a Y582F mutation had milder effects. In fluorescence anisotropy experiments, the Y582F mutant showed slightly lower UNC-45 affinity compared to the native peptide (Fig. 5a), while in the cellular chaperone assay, the amounts of soluble myosin were comparable to that of wildtype protein (Fig. 5c). Consistently, C. elegans non-muscle myosins contain a Phe582, suggesting that the phenyl ring at this position is sufficient for UNC-45 assisted folding (Supp. Fig 8b). Taken together, our in vitro and in vivo data highlight the importance of the conserved FX 3 HY motif to mediate interactions with UNC-45 and promote myosin folding. Moreover, our findings directly connect the Y582S mutation found in FSS myopathy to myosin quality control, preventing chaperone binding thus interfering with myosin maturation and causing aggregation of the muscle protein.
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Discussion
Structural characterization of chaperone-substrate interactions is challenging due to the heterogeneity of respective complexes, which contains a continuum of transient folding intermediates. Advances in cryo-electron microscopy and NMR spectroscopy allowed visualization of certain chaperone-substrate complexes 26,[35][36][37][38][39][40][41][42][43] , delineating the targeting mechanisms of general chaperones including HSP90, HSP70 and TriC 26,[35][36][37][38][39][40][41][42][43] . Conversely, the mode of action of the many client-specific folding factors is less understood, with notable exceptions being the histone and Rubisco chaperones 44,45 . To better understand substrate targeting by specialized chaperones, we studied the interplay between UNC-45 and its client protein myosin. By applying an integrative structural biology approach, we show that the highly conserved FX 3 HY motif present in the myosin motor domain is a discrete molecular epitope recognized by the UNC-45 chaperone, which then presents the bound client to downstream PQC factors.
An important feature of the PQC decision-making process is that UNC-45 can accommodate both folded and misfolded myosin. Upon forming separate complexes with the two myosin forms, downstream acting chaperones and E3 ligases channel the presented client into folding or degradation pathways (Fig. 6). Whereas the general chaperones, as shown here for HSP90, seem to obtain an indirect role preventing premature myosin aggregation and keeping the (partially) misfolded protein in solution, the UFD-2 ubiquitin ligase takes the decision. Only the severely damaged myosin is targeted whereas folded myosin in complex with UNC-45 is channeled into assembly pathways. Importantly, for myosin ubiquitination by UFD-2, formation of a productive E3 ubiquitin ligase complex depends on a composite signal, The stable complex of UNC-45:myo F with predominantly folded myosin implies that UNC-45 not only mediates the initial stages of protein folding, but also possesses a function beyond, a finding that is compatible with the role of UNC-45 in thick filament assembly and stress response 13,21 . Chaperone complexes, which engage with folded substrates, have also been observed in other biological pathways. For example, a . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 9, 2022. ; https://doi.org/10.1101/2022.08.08.502945 doi: bioRxiv preprint 13 specific J-domain protein was shown to bind natively folded tau in order to prevent protein aggregation 46 . Intriguingly, Tyr582 of the myosin FX 3 HY-motif has to fulfill two critical functions. First, Tyr582 has been shown to promote structural rearrangements in the myosin motor required for the ATP-driven power stroke 47,48 . In addition, as shown here, Tyr582 is one of three conserved myosin residues that is essential for UNC-45 binding. Given the importance of Tyr582 for motor activity and myosin folding, our findings suggest that binding of UNC-45 to myosin could have a regulatory role, coupling chaperone engagement with inhibition of ATPase activity.
This regulation might be critical to keep the myosin motor in a resting state during protein folding, sarcomere assembly or under stress conditions. Consistently, a previous study highlighted the role of UNC-45 in inhibiting the myosin power stroke, and suggested that inhibition is achieved through interactions with the UCS domain 49 .
The respective interplay between UNC-45 and myosin is now resolved in molecular detail, revealing a highly specific binding mode tailored to target a key functional motif of the client. Other client-specific chaperones are likely to also target conserved functional sites in their substrates, ensuring a high selective pressure to maintain specialized folding pathways.
Myosin-linked myopathies have been mostly studied in the light of deregulated myosin ATPase activity that impairs the actomyosin cross-bridge cycle 50 or the energy-saving super-relaxed state (SRX) 51,52 . Interestingly, mutations in chaperones like Bag3 53 and DnaJB6 54 cause congenital muscle defects, and HSPB6, HSP70 and HSP90 are upregulated upon cardiac failure 55,56 , pointing to the importance of an efficient PQC system to prevent myosin-related myopathies. Consistently, human UNC-45B mutations have been linked to mild forms of myopathy, attributed to reduced stability of the chaperone itself 17 . The characterized mutations are localized in the UCS domain, close to residues identified as myosin interaction sites (Supp. Fig. 9), suggesting that they might impact not only UNC-45 stability, but also substrate interactions. We thus propose that myosin proteostasis, and specifically UNC-45:myosin interactions, play an important yet underestimated role in myopathies. The importance of a functional PQC system for preventing human myopathies is reflected by the Y582S mutation in the FX 3 HY motif, underlying FSS developmental myopathy 34 . This point mutation abolishes the interaction with UNC-45, leading to myosin misfolding and aggregation. To our knowledge, these findings provide the first direct link between impaired myosin quality control and the onset of . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 9, 2022. ; https://doi.org/10.1101/2022.08.08.502945 doi: bioRxiv preprint 14 myopathies, offering new paths for medical intervention. Moreover, our data indicate that disease causing mutations in myosin and UNC-45 are recapitulated by the C. elegans proteins. Given its powerful genetics and emerging role to study protein misfolded diseases 57-59 , C. elegans should be an attractive model system to delineate cellular pathways causing myosin-based myopathies and explore counteractive strategies.

Declaration of interests
The authors declare no competing interests.
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Isothermal titration calorimetry (ITC)
For peptide quantification a tryptophan was introduced at the N-terminus. This residue is visible in the SHE4-myosin co-crystal structure but was omitted from the discussion since it is not part of the native protein sequence. ITC for characterizing interactions between SHE4 and the synthesized myosin peptides was performed at 25 °C with a VP-ITC (Microcal). Peptides and protein were diluted in 10 mM HEPES

Protein sequence analysis
For conservation analysis of the UCS-domain representative proteins from the UNC-45, CRO1 and SHE4 family were selected. For analysis of myosin conservation, all annotated myosin sequences from the UniProt repository were used. Multiple sequence alignment was performed with MUSCLE 64 and conservation was plotted using Jalview 65 applying the BLOSUM62-score.

Peptide Spot assays
Myosin-peptides (MHC-B, C. elegans, residues 1-790) consisting of 15 amino acids were synthesized on a derivatized cellulose membrane (Intavis 32.100) using the ResPep SLi synthesizer (Intavis). The membrane was designed with an overlap of 12 amino acid residues compared to the neighboring peptide. The membrane was . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 9, 2022. ; https://doi.org/10.1101/2022.08.08.502945 doi: bioRxiv preprint 28 activated with methanol, blocked with 3% bovine serum albumin (BSA) and incubated with 50 μ g/ml Strep-tagged (C-term) wildtype UNC-45/UNC-45ΔUCS/UNC-45Δloop.
The peptide spot membrane was blotted onto a nitrocellulose membrane and UNC-45 binding was probed with an anti-Strep antibody (Qiagen) using an ECL-kit for detection (GE Healthcare).

Thermal shift assays
These assays were performed in 96-well-plate format using a RT-PCR thermocycler  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 9, 2022. ; https://doi.org/10.1101/2022.08.08.502945 doi: bioRxiv preprint 29 When purified UNC-45:myosin-complexes were used as substrate, 1 μ M of the indicated SEC-fractions were subjected to the ubiquitination assay after purification without prior incubation. Ubiquitination assays were performed at 21 °C, at the respective timepoints the reaction was stopped by adding SDS-PAGE loading buffer and heating the sample to 95 °C for 5 minutes. Samples were electrophoretically separated on BioRad Stain-Free TGX 4-20% gels. Proteins modified with ubiquitin were visualized by DyLight800-fluorescence and total protein levels were measured using Stain-Free tryptophan fluorescence. Signals were quantified using Fiji software. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 9, 2022. ; https://doi.org/10.1101/2022.08.08.502945 doi: bioRxiv preprint 30 5% (v/v) acetonitrile, 0.1% (v/v) TFA. The nano HPLC system used was an UltiMate 3000 RSLC nano system (Thermo Fisher Scientific) coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific), equipped with a Proxeon nanospray source (Thermo Fisher Scientific). Peptides were loaded onto a trap column at a flow rate of 25 μ l/min using 0.1% TFA as mobile phase. After 10 min, the trap column was switched in line with the analytical column (both columns from Thermo Fisher Scientific). Peptides were eluted at a flow rate of 230 nl/min, and a binary 3 h gradient. The gradient started with 98% mobile phase A (water/formic acid, 99.9%/0.1%, v/v) and 2% mobile phase B (water/acetonitrile/formic acid, 19.92%/80%/0.08%, v/v/v), increased to 35% mobile phase B over 180 min, followed by a 5 min-gradient to 90% mobile phase B. Acquisition was performed in datadependent mode with a 3 s cycle time. The full scan spectrum was recorded at a resolution of 60 000 in the range of 350-1500 m/z. Precursors with a charge state of +3 to +7 were fragmented. HCD-collision energy was set to 29%. The resolution of MS2-scans recorded in the Orbitrap was 45 000 with a precursor isolation width of 1.0 m/z. Dynamic exclusion was enabled with 30 s exclusion time.
Fragment spectra peak lists were generated from the raw MS-data using the software MSConvert 67 (v 3.0.20105) selecting the peak picking filter. Crosslink search was performed using XiSearch 68 (v 1.7.4) applying the following parameters: 6 ppm MS1accuracy; 20 ppm MS2-accuracy; DSS-crosslinker with reaction specificity for lysine, serine, threonine, tyrosine and protein N-termini with a penalty of 0.2 (scale 0-1) assigned for serine, threonine and tyrosine; carbamidomethylation of cysteine as a fixed modification; oxidation of methionine as variable modification; tryptic digest with up to four missed cleavages; all other variables were used at default settings.
Identified crosslinks were filtered to 5% FDR on link level with the software XiFDR 69 (v 1.4.3.1). For plotting of crosslink data, the in-house software CrossLinkingVisualizer was used 70 .

Solubility assay
Hi5 cells were infected for 4 days at 21°C with a baculovirus expressing both UNC-45 WT and myosin variants in the same backbone, as for the "Protein expression" section. Cells were then spinned at 1000G for 15 minutes, weighted and flash-frozen in liquid nitrogen. The pellets were then resuspended in 50 mM Na Phospate pH 8, 300 mM NaCl, 1:5000 benzonase. The volume of buffer for the resuspension was . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 9, 2022. ; https://doi.org/10.1101/2022.08.08.502945 doi: bioRxiv preprint 31 proportional to the weight of the pellet (5X). The suspension was centrifuged at 20000G for 30 minutes to separate soluble and insoluble fractions. The supernatant of the clear cell lysate was then loaded on a 10% self-cast SDS-PAGE. Quantification of the band intensity was performed with ImageJ, and plotting and statistical analysis (paired t-test) was performed with GraphPad prism.
. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 9, 2022. ; https://doi.org/10.1101/2022.08.08.502945 doi: bioRxiv preprint